Myosin-binding protein C (MyBP-C) is a thick (myosin) filament component of vertebrate striated muscle that plays a key role in modulating contraction. Three distinct isoforms are encoded by different genes, resulting in the expression of fast and slow skeletal muscle MyBP-C isoforms and a third (cardiac) isoform. Since its discovery in skeletal muscle 40 years ago, most studies of MyBP-C have focused on the cardiac isoform, because mutations in this isoform are a prime cause of inherited cardiomyopathies. However, the recent discovery that mutations in slow skeletal MyBP-C cause skeletal muscle myopathies, one of which is neonatally lethal, makes it clear that defining the molecular structure and function of the skeletal MyBP-C isoforms is critically important. Therefore, in this dual-PI proposal, PIs Craig (UMMS) and Warshaw (UVM), in collaboration with Drs. Irving (Illinois) and Sadayappan (Loyola), will combine their labs' expertise in high resolution imaging and single molecule biophysics coupled with X-ray diffraction, molecular biology and mass spectrometry to elucidate the molecular structure and function of skeletal MyBP-C. In Aim 1, in situ and in vitro model systems will help determine if MyBP-C activates and/or mechanically modulates the calcium- dependent sliding of native thin (actin) filaments over native thick filaments from fast and slow rat skeletal fibers and whether contractile modulation occurs only where MyBP-C exists in the thick filament. In Aim 2, through a novel super-resolution light microscopic technique, we will determine whether the MyBP-C N terminus functions by binding to actin and/or myosin. In complementary experiments, fiber X-ray analysis and EM 3D reconstruction of native thin and thick filaments will determine if MyBP-C displaces tropomyosin to activate the thin filament and/or directly influences myosin head interactions to modulate head function. In Aim 3, the structural and functional consequences of MyBP-C N-terminal domain isoform differences between fast and slow MyBP-C will be characterized with special emphasis on 2 slow MyBP-C splice variants thought to affect actin and myosin binding. Through structural mutagenesis, N-terminal fragments will be expressed with domain deletions and slow MyBP-C splice inserts in an effort to define the domains and inserts that confer MyBP-C's modulation of actomyosin function. Although skeletal MyBP-C's clinical impact is apparent, its functional role is far from certain and thus this dual-PI proposal, tightly integrating MyBP-C structure and function, offers an opportunity to rapidly advance our understanding of both fast and slow skeletal MyBP-C isoforms in their normal state.